1. Field of the Invention
This invention relates to processes for preparing nitroaromatic glycosides, particularly nitroaromatic derivatives of maltotetraose, maltopentaose, and maltohexaose, useful as standard substrates for the assay of .alpha.-amylase in serum and other biological liquids.
2. Relation to the Prior Art
1. U.S. Pat. No. 3,879,263, issued Apr. 22, 1975 discloses the determination of the .alpha.-amylase content of biological samples by adding maltotetraose or maltopentaose to the sample at constant temperature and pH. The process allows rapid determination of .alpha.-amylase, and can be used to differentiate between saliva .alpha.-amylase and pancreas .alpha.-amylase. The latter produces glucose, whereas the former does not. The glucose produced may be estimated spectrophotometrically, e.g., by nicotinamide-adenine dinucleotide (reduced form) (NADH) absorption of 340 nm. Because this assay depends upon glucose, a glucose detecting reaction is necessary. Furthermore, if glucose is present in the sample, it must either be removed or compensated for. The compounds of the present invention differ from this in that 4-nitrophenol is released as the substance which can then be related to .alpha.-amylase. This makes the assay independent of the glucose detecting step.
2. A. P. Jansen and P. G. A. B. Wydeveld, Nature, 182, 525 (1958) postulate that .alpha.-(p-nitrophenyl)maltoside could be a substrate for an amylase assay. However, this paper shows that the authors never identified the active agent responsible for their observations. They reported: (1) Incubation of human urine or saliva samples with .alpha.-(p-nitrophenyl)maltoside at 37.degree. for 16 hr produced 4-nitrophenol, identified spectrophotometrically by mixing the hydrolyzate with 0.02 N sodium hydroxide. (2) The hydrolysis was inhibited by protein precipitants such as 10% trichloroacetic acid and 0.5 N silver nitrate. (3) The hydrolysis was pH-dependent, being most effective at pH 5.9-7.0. They state that this was evidence for "the possible existence of an unidentified carbohydrase". .alpha.-(4-Nitrophenyl)maltoside is not believed to be useful for an amylase assay because the cleavage of this compound by .alpha.-amylase is extremely slow.